ABSTRACTFibroblast growth factor-1 (FGF1) and FGF2 play a critical role in angiogenesis, a formation of new blood vessels from existing blood vessels. Integrins are critically involved in FGF signaling through crosstalk. We previously reported that FGF1 directly binds to integrin αvβ3 and induces FGF receptor-1 (FGFR1)-FGF1-integrin αvβ3 ternary complex. We previously generated an integrin binding defective FGF1 mutant (Arg-50 to Glu, R50E). R50E is defective in inducing ternary complex formation, cell proliferation, and cell migration, and suppresses FGF signaling induced by WT FGF1 (a dominant-negative effect) in vitro. These findings suggest that FGFR and αvβ3 crosstalk through direct integrin binding to FGF, and that R50E acts as an antagonist to FGFR. We studied if R50E suppresses tumorigenesis and angiogenesis. Here we describe that R50E suppressed tumor growth in vivo while WT FGF1 enhanced it using cancer cells that stably express WT FGF1 or R50E. Since R50E did not affect proliferation of cancer cells in vitro, we hypothesized that R50E suppressed tumorigenesis indirectly through suppressing angiogenesis. We thus studied the effect of R50E on angiogenesis in several angiogenesis models. We found that excess R50E suppressed FGF1-induced migration and tube formation of endothelial cells, FGF1-induced angiogenesis in matrigel plug assays, and the outgrowth of cells in aorta ring assays. Excess R50E suppressed FGF1-induced angiogenesis in chick embryo chorioallantoic membrane (CAM) assays. Interestingly, excess R50E suppressed FGF2-induced angiogenesis in CAM assays as well, suggesting that R50E may uniquely suppress signaling from other members of the FGF family. Taken together, our results suggest that R50E suppresses angiogenesis induced by FGF1 or FGF2, and thereby indirectly suppresses tumorigenesis, in addition to its possible direct effect on tumor cell proliferation in vivo. We propose that R50E has potential as an anti-cancer and anti-angiogenesis therapeutic agent ("FGF1 decoy").

Mentions:
CAM is another widely utilized in vivo system to study angiogenesis and anti-angiogenesis and it is easier to quantify angiogenesis in this assay than other assays. We placed saline- or FGF- impregnated filter disks on blood vessels in avascular sections of CAM (day 11) for 48 h to induce angiogenesis. The disks and underlying CAM tissue (day 13) were then harvested. We scored angiogenesis by counting vessel branches present in the CAM tissue below the filter from digital images. We first determined optimum dose of wt FGF1 for angiogenesis (Fig. 6a, 6b). Five ng/ml of wt FGF1 was optimum. R50E (5 and 50 ng/ml) did not induce angiogenesis. We tested if excess R50E (50 ng/ml) suppresses angiogenesis induced by WT FGF1 (5 ng/ml). Notably, excess R50E suppressed angiogenesis induced by WT FGF1 (Fig. 6c). This suggests that R50E shows an anti-angiogenic action in this model as well. Since FGF1 binds to all known FGFRs (FGFR1-4), R50E is expected to suppress FGFR signaling induced by other members of the FGF family. We tested if R50E suppresses angiogenesis induced by FGF2. We found that this is the case: excess R50E suppressed angiogenesis induced by WT FGF2 (Fig. 6c). The data suggest that R50E suppresses FGF1- and FGF2-induced angiogenesis in the CAM model.

Mentions:
CAM is another widely utilized in vivo system to study angiogenesis and anti-angiogenesis and it is easier to quantify angiogenesis in this assay than other assays. We placed saline- or FGF- impregnated filter disks on blood vessels in avascular sections of CAM (day 11) for 48 h to induce angiogenesis. The disks and underlying CAM tissue (day 13) were then harvested. We scored angiogenesis by counting vessel branches present in the CAM tissue below the filter from digital images. We first determined optimum dose of wt FGF1 for angiogenesis (Fig. 6a, 6b). Five ng/ml of wt FGF1 was optimum. R50E (5 and 50 ng/ml) did not induce angiogenesis. We tested if excess R50E (50 ng/ml) suppresses angiogenesis induced by WT FGF1 (5 ng/ml). Notably, excess R50E suppressed angiogenesis induced by WT FGF1 (Fig. 6c). This suggests that R50E shows an anti-angiogenic action in this model as well. Since FGF1 binds to all known FGFRs (FGFR1-4), R50E is expected to suppress FGFR signaling induced by other members of the FGF family. We tested if R50E suppresses angiogenesis induced by FGF2. We found that this is the case: excess R50E suppressed angiogenesis induced by WT FGF2 (Fig. 6c). The data suggest that R50E suppresses FGF1- and FGF2-induced angiogenesis in the CAM model.

ABSTRACTFibroblast growth factor-1 (FGF1) and FGF2 play a critical role in angiogenesis, a formation of new blood vessels from existing blood vessels. Integrins are critically involved in FGF signaling through crosstalk. We previously reported that FGF1 directly binds to integrin αvβ3 and induces FGF receptor-1 (FGFR1)-FGF1-integrin αvβ3 ternary complex. We previously generated an integrin binding defective FGF1 mutant (Arg-50 to Glu, R50E). R50E is defective in inducing ternary complex formation, cell proliferation, and cell migration, and suppresses FGF signaling induced by WT FGF1 (a dominant-negative effect) in vitro. These findings suggest that FGFR and αvβ3 crosstalk through direct integrin binding to FGF, and that R50E acts as an antagonist to FGFR. We studied if R50E suppresses tumorigenesis and angiogenesis. Here we describe that R50E suppressed tumor growth in vivo while WT FGF1 enhanced it using cancer cells that stably express WT FGF1 or R50E. Since R50E did not affect proliferation of cancer cells in vitro, we hypothesized that R50E suppressed tumorigenesis indirectly through suppressing angiogenesis. We thus studied the effect of R50E on angiogenesis in several angiogenesis models. We found that excess R50E suppressed FGF1-induced migration and tube formation of endothelial cells, FGF1-induced angiogenesis in matrigel plug assays, and the outgrowth of cells in aorta ring assays. Excess R50E suppressed FGF1-induced angiogenesis in chick embryo chorioallantoic membrane (CAM) assays. Interestingly, excess R50E suppressed FGF2-induced angiogenesis in CAM assays as well, suggesting that R50E may uniquely suppress signaling from other members of the FGF family. Taken together, our results suggest that R50E suppresses angiogenesis induced by FGF1 or FGF2, and thereby indirectly suppresses tumorigenesis, in addition to its possible direct effect on tumor cell proliferation in vivo. We propose that R50E has potential as an anti-cancer and anti-angiogenesis therapeutic agent ("FGF1 decoy").